Osteosclerosis is a major complication of primary myelofibrosis (PMF) and is characterized, in addition, by excess proliferation of bone marrow myeloid cells, abnormal connective tissue and fibrosis. Although mutant megakaryocytes (MKs) play a major role in driving connective tissue abnormalities in PMF, molecular mediators which develop between abundant MKs in PMF and increased bone formation from mesenchymal progenitors have not been established. Here, we propose to examine an innovative concept involving the multifunctional enzyme known as lysyl oxidase (LOX) and other MK-derived growth factors as agents which directly promote osteosclerosis in PMF by stimulating the proliferation and differentiation of bone marrow mesenchymal stem cells (MSCs). Human PMF is most often caused by clonal expansion promoted by a hyper- activating causative mutation in the JAK2 gene. In Aim 1 we propose to use a mouse model of JAK2V617F? induced PMF to test the new contention that factors elaborated by JAK2V617F MKs impact bone marrow MSCs differentiation to osteoblasts. To this end, JAK2V617F MKs will be tested in co-cultures with wild type MSCs. Based on preliminary and other studies, levels of candidate factors (LOX, PDGF, TGF?, WNT3a) will first be assessed by ELISA assays, and functionality determined with blocking antibodies and proteins, and by LOX pharmacological inhibition. In the spirit of a relatively higher risk R21 grant mechanism, we also propose to measure 110 different cytokines via state-of-the-art Luminex assays, followed by blocking experiments of differentially regulated factors. In Aim 2, we will examine the novel hypothesis that LOX contributes to the pathological bone phenotype in PMF in vivo. To assess this, we will cross breed JAK2V617F mice with LOX+/- mice available to us, and then characterize the bone phenotype of compound mutant mice JAK2V617F/LOX+/- in vivo and in vitro. Taken together, data obtained in this project will determine factors elaborated by associations between JAK2V617F MKs and wild type MSCs which drive osteosclerosis, and which, therefore, will become potential therapeutic targets to address osteosclerosis in PMF.